Method for separating non-high density lipoproteins from lipoprotein containing body fluid samples

ABSTRACT

The invention relates to a method useful in separating non-high density lipoproteins, referred to as &#34;non-HDLs&#34;, from biological fluids containing them. A porous carrier is provided which contains a non-HDL precipitating agent. One need only contact the sample of interest to the carrier for one minute or less, after which the precipitated non-HDL-s are no longer present in the sample being tested. The applications of the method include the ability to determine high density lipoproteins in the sample without interference from non-HDLs.

This is a Divisional Application under 37 CFR 1.60 of application Ser.No. 08/384,046 filed on Feb. 6, 1995, now U.S. Pat. No. 5,580,743, whichapplication was a Divisional Application under 37 CFR 1.60 ofapplication Ser. No. 07/572,875, filed Aug. 24, 1990, now U.S. Pat. No.5,426,030.

The invention concerns a method for the quantitative determination ofHDL (high Density Lipoprotein) in biological fluids and an agentsuitable therefor.

Total cholesterol in blood, plasma or serum is one of the best knownparameters for assessing the extent of risk of a coronary heart disease.However, the concentration of total cholesterol is only of limited valuefor the assessment of individual risk. The measurement of thecholesterol in the lipoproteins of low density (Low densityLipoproteins=LDL) on the one hand and in the lipoproteins of highdensity (High Density Lipoproteins=HDL) on the other hand is moremeaningful. Epidemiological and clinical studies have shown that thereis a positive correlation between LDL cholesterol and coronary heartdisease and a negative correlation between HDL cholesterol and coronaryheart disease.

As a close approximation the determination of the HDL as well as thetotal cholesterol is sufficient for an assessment of risk. This courseis preferably followed at present in diagnostic practice.

The other lipoprotein classes (LDL, VLDL, chylomicrons) which arepresent have to be separated in order to determine HDL cholesterolseparately. The potential methods of separation are based on differencesin the flotation densities (sequential flotation or equilibriumsedimentation, both in the ultracentrifuge), on different surfacecharges (electrophoreses on paper or agarose as carrier) or ondifferences in the apolipoproteins (immunochemical methods usingspecific antibodies). All these methods of separation are expensive,time-consuming and not established in routine laboratories.Precipitation reactions (in Monographs on Atherosclerosis, Vol. 11(1982), Clackson, T. B., Kritchevsky, D., Pollak, O. J. eds; LipoproteinPrecipitation, Burstein, M., Legmann, P. and in Meth. in Enzymology,Vol. 129 (1986)) whose specificity depends on the particle dimension andthe surface charge are cheap, relatively easy to handle and thereforewidespread. Polymeric substances serve as precipitation reagents, andthese are usually polyanionic. Polymers which are uncharged are alsosuitable. The polyanions usually need bivalent cations in order todevelop their precipitating effect, while the uncharged polymers do notrequire them.

The experimental procedures and the concentrations which are used forthe combined precipitation agents are designed to quantitativelyprecipitate all lipoproteins except HDL, to separate these precipitatesfrom the liquid fraction of the sample in a suitable manner andsubsequently to quantify the HDL in the liquid fraction of the sample bymeans of a cholesterol assay. For this, depending on how the test iscarried out, a defined volume of precipitating agent (in suitableconcentrations) is mixed intensively with a defined volume of the sampleto be determined. It is the state of the art to allow a reaction time ofat least 10 minutes for the precipitation and only after this timeinterval has elapsed to separate sedimented non-HDL lipoproteinprecipitates and HDL remaining in the liquid fraction by centrifugation.The centrifugation step also needs some time.

This method is much too time-consuming for a routine test. In addition,centrifugation steps with subsequent separation of the supernatant needcomplicated additional equipment and a transfer step. The requiredpipetting procedure in which a defined amount of supernatant is takenis, in addition, a source of error which can lead to less precisemeasurements.

It is therefore the object of the invention to avoid the disadvantagesof the state of the art and to provide a method for the separation ofnon-HDL lipoproteins from biological fluids, which can be carried outmore rapidly and without complicated additional equipment and whichallows a more rapid and a simpler HDL cholesterol determination.

The object is achieved by a method for the separation of non-HDLlipoproteins from biological fluids, in which the biological fluidcontaining non-HDL lipoproteins is applied onto a carrier through whichliquids can flow and which contains α-precipitating agent for non-HDLlipoproteins. The invention also provides an agent for the separation ofnon-HDL lipoproteins and a rapid diagnostic agent which contains thisagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a cross-section showing the layers of a test strip inaccordance with a method of the invention.

FIG. 2 represents a side view of the test strip.

It was found that the precipitation of the non-HDL lipoproteins proceedsparticularly rapidly and specifically with conventional, well-knownprecipitating agents when these precipitating agents are applied in afinely dispersed form onto a carrier through which liquids can flow andthe sample has to flow through this carrier. In general, theprecipitation takes less than 1 min. Carriers through which liquids canflow are: papers, fabrics made of synthetic fibres such as e.g.polyester or polyamide or others, fabrics made of natural fibres such ascotton, silk or others or mixtures of these materials. In thisconnection, the structure of the fabrics can be monofilament ormultifilament, multifilament forms being preferred.

The fibres which compose the carrier through which liquids can flowpreferably have a fibre diameter from 3 to 100 μm, preferably 5 to 50μm. The carrier has in particular a weight per unit area of 10 to 100g/m², preferably 10 to 50 g/m² at a thickness of 0.030 to 0.150 mm, andthe ability to absorb water is 25 to 100 g/m², preferably 40 to 70 g/m²at the thicknesses mentioned above.

Other suitable carriers are arrangements of glass fibres and mixtures ofglass fibres with the fibres mentioned above through which liquids canflow--preferably in the form of fleece--and membranes in differentforms. The membranes should have hydrophilic properties, a thicknessbetween 20-250 μm, preferably 70-150 μm and pore sizes between 0.2-20μm, preferably 5-15 μm.

The transport of liquid in these carriers is based on capillary forces.

The precipitating agents are preferably applied to the carrier byimpregnating the carrier with a solution, emulsion or suspension of theprecipitating agent and subsequent drying. In this process other usefuladditives such as pH buffer substances or detergents etc. can also beapplied.

In this construction all chemical compounds are suitable asprecipitating agents which can also be used in a "wet chemical" processfor the precipitation of lipoproteins as long as they dissolve quicklyenough in the sample solution. Certain polyanions in combination withbivalent cations are in particular known. Examples of these includecombinations of phosphotungstic acid and magnesium chloride, of heparinand manganese (II) chloride or of dextran sulphate and magnesiumchloride. It should be noted that, in principle, each polyanion can becombined with each of the three cations (Mg²⁺, or Mn²⁺, or Ca²⁺) whichresults, however, in slight differences in their capacity to precipitatelipoproteins (Burstein, 1986). The concentration of the chosen cationcan be adapted accordingly in order to specifically precipitate thenon-HDL lipoproteins. The molecular size of the polyanion used alsoinfluences the capacity to precipitate lipoproteins and should be takeninto account when choosing the concentrations. The use of dextransulphate with a molecular weight of 500000 and 50000, for example, isknown. Both are also suitable for an application on carriers throughwhich liquids can flow. Dextran sulphate with a molecular weight of50000 combined with Mg²⁺ is, however, preferred in which case Mg²⁺ ispreferably used in the form of magnesium acetate. In principle,magnesium sulphate, magnesium chloride and magnesium aspartate can, alsobe used.

The concentration of the precipitating agent can be matched exactly tothe volume of the sample to be examined.

The non-HDL lipoproteins are precipitated by bringing a sample intocontact with the carrier through which liquids can flow and whichcontains the precipitating agent. The precipitation reaction is startedthereby. The time interval during which the sample is in contact withthe precipitating agent on the precipitating agent carrier can beadjusted in a simple manner by varying the suction pressure by means ofthe interstitial volumes or the hydrophilicity of the carrier. Theachieved flow rates of the sample through the carrier are very importantfor the test performance. The precipitate formation is completed afterless than one minute, at best even after ca 10 sec and the liquid can beremoved from the carrier material. This can be effected continously ordiscontinously, for example by decanting, using gravitational force orby aspirating. The fluid is preferably drawn into a further carrier bycapillary forces in which the precipitated non-HDL lipoproteins areseparated. This process requires that the carriers are in contact withone another. The separation of the precipitates by other methods such ascentrifugation is less preferable even though this is also possiblewithout losing the advantage of the fast precipitation.

It was found that lipoprotein precipitates which are formed by theaction of a combination of polyanions and the bivalent cations mentionedabove on biological samples can be retained in a mesh of fibres.

The arrangement of the fibres in the mesh is preferably disordered.Glass fibres, cellulose fibres, polyamide fibres and polyester fibres ormixtures thereof are preferably used as the fibres. Glass fibres andmixtures with the fibres mentioned above are particularly preferred. Thefibres which make up the mesh preferably have a diameter of 0.2 to 10.0μm, preferably a diameter of 0.5 to 5.0 μm. The mesh has a weight perunit area of 20 to 50 g/m², preferably 23 to 30 g/m² and a capacity toabsorb water of 250 to 500 g/m², preferably of 320 to 420 g/m² at athickness of 0.19 to 0.23 mm.

Systems containing glass fibres, their possible spatial arrangement andthe dimension of the fibres are described in detail in DE 30 29 579.Furthermore, the mesh of fibres can be hardened by the addition ofsuitable binding agents, either of an inorganic nature (e.g. waterglass) or organic nature (e.g. polymers such as polyvinyl acetate,polyacrylic acid ester or similar polymers). These additives inter aliaagglutinate the fibres at those positions where they are in contact withone another and in this way they improve the mechanical stability of thefibre mesh.

The liquid fraction of the applied sample spreads unhindered through thewhole mesh and it can also leave it when the geometric arrangement isapproximately arranged or configured or by overcoming the internalcapillary forces of the mesh. In this way the separation of precipitatednon-HDL lipoproteins and non-precipitated HDL can be achieved withsimple devices. Biological fluids from which the non-HDL lipoproteinscan be separated according to the present invention include whole blood,serum and plasma.

The method can be particularly advantageously used in methods for thequantitative determination of HDL cholesterol on test strips. For this,the fluid from which the non-HDL lipoproteins have been separatedaccording to the method described above, is brought into contact withreagents which are necessary and/or useful for carrying out the testreaction, for example a test reaction for cholesterol. Such reagents areknown to the expert, for example from EP-B-0016387, and can be adaptedto the expected cholesterol concentrations (0-100 mg/dl). They can forexample, also be present in the form of a film or a coating on a porouscarrier. Such embodiments of reagent forms are known to the man skilledin the art.

An embodiment of a method for the determination of HDL cholesterol whichcontains the preferred embodiment for separating the non-HDLlipoproteins is shown in FIG. 1. A rapid diagnostic agent (1) containsseveral layers of a carrier in a casing (2).

A fibre mesh (5) with a separating capacity for lipoprotein precipitateswhich is adjusted to the chosen sample volume is underneath a carrierthrough which liquids can flow and which is impregnated with aprecipitating agent (4). Adjoining this is a suitable absorptivecholesterol determining test film (6) which is capable of taking upfluid free of precipitate from the fibre mesh. The test film is coatedon a transparent foil (7). The determination is started by applying thesample liquid over the carrier containing the precipitating agent. Themeasurement signal which develops can be evaluated visually orphotometrically from the foil side of the construction.

This simple design is not well-suited for the investigation of wholeblood. If whole blood is to be used, then the cellular blood componentsare preferably separated in a layer (3) in front of the carrier for theprecipitating agent e.g. by agents which are described in detail inDE-A-30 29 579.

With regard to the well-known temperature dependency of enzymaticreactions, the geometric arrangement of the test design can also be sochosen that the cholesterol test is separated by time from the previousreaction steps. This also allows a controlled regulation of thetemperature of the test step. A corresponding test construction isoutlined in DE-A-3130749. The preferred construction of a rapiddiagnostic agent for HDL in a test strip form (11) is shown in FIG. 2. Afibre mesh (15), a carrier through which liquids can flow and whichcontains a precipitating agent (14) as well as a separating layer forcellular components (13) are mounted on top of one another on asupporting foil (12). The fibre mesh (15) protrudes from under thecarriers (13) and (14) towards a flap. The flap, which consists of atransparent foil (17) and a test film for cholesterol (16) is attachedto the supporting foil (12) via an adhesive join (18).

The sample to be investigated is applied to the layer (13) and flowsthrough the layers (14) and (15), during which the non-HDL lipoproteinsare separated, into the mesh (15) under the flap. The test reaction isstarted by pressing the flap with the test layer (16) onto the layer(15). The change in colour can be followed by means of a photometer or areflection photometer.

In this case in comparison with the embodiment outlined in FIG. 1lateral separating capacity of the fibre mesh is also effective.

The method according to the present invention has other major advantagesover the known methods. It is possible to also use particularly smallliquid volumes. The handling of the device according to the presentinvention is very simple. Only two basic handling steps are necessary,namely the application of a liquid sample and the reading of ameasurement after a particular time. Other instruments are not neededapart from a suitable photometer in the case of a quantitativedetermination. Transfer steps are not needed. All types of blood, evenwhole blood, can be easily used when a separating pad for cells is usedfirst. If anticoagulants have been added to the sample to be examined,it is recommended that their effects on the determination becompensated. The separation of non-HDL lipoproteins can be carried outin less than 60 sec. The dosage of the sample volume is greatlysimplified.

Examples for the invention are given in the following:

EXAMPLE 1

Precipitating agent carrier (14 or 4) for EDTA Plasma

A multifilament polyester fabric (100 μm mesh size, 105 μm fabricthickness, 55 threads per cm, with 815 l water passage per m² and sec)is impregnated with a solution of the following composition:

    ______________________________________                                        Hepes buffer, 50 mM; pH 7.0                                                                           78.00  g                                              magnesium acetate × 4H.sub.2 O                                                                  15.68  g                                              dextran sulphate (MW 50000)                                                                           2.57   g                                              bovine serum albumin    1.78   g                                              ______________________________________                                    

A coating of ca 57 ml/m² is obtained.

After drying under a flow of warm air the impregnated fabric is cut intosuitable unit areas which match the corresponding test procedure andthey are integrated into the test construction.

EXAMPLE 2

Precipitating agent carrier (14 or 4) for serum

The following impregnating solution is used in the procedure analogousto Example 1:

    ______________________________________                                        Hepes buffer, 50 mM; pH 7.0                                                                           78.00  g                                              magnesium acetate × 4H.sub.2 O                                                                  10.10  g                                              dextran sulphate (MW 50000)                                                                           2.57   g                                              bovine serum albmin     1.78   g                                              H.sub.2 O               5.58   g                                              ______________________________________                                    

EXAMPLE 3

Precipitating agent carrier (14 or 4) for blood

A paper of suitable thickness and absorptivity e.g. tea bag paper with aweight per unit area of 12 g/cm² and a thickness of 50 μm is impregnatedwith the following impregnating solution:

    ______________________________________                                        Hepes buffer, 50 mM; pH 7.0                                                                           70.20  g                                              magnesium acetate × 4H.sub.2 O                                                                  4.78   g                                              dextran sulphate (MW 50000)                                                                           2.54   g                                              bovine serum albumin    1.60   g                                              H.sub.2 O               7.80   g                                              ______________________________________                                    

A coating of ca. 63 ml/m² is obtained with this carrier.

EXAMPLE 4

Test film

A dispersion of the following composition:

    ______________________________________                                        K/Na phosphate buffer, 0.5 M; pH 7.0                                                                  17.14   g                                             Keltrol F               0.19    g                                             TiO.sub.2  (powder)     1.31    g                                             dioctyl sodium sulphosuccinate                                                                        0.40    g                                             polyvinylpropionate dispersion                                                                        11.70   g                                             (50% in H.sub.2 O)                                                            diatomaceous earth (Celatom MW 25)                                                                    17.65   g                                             phenylsemicarbazide     0.025   g                                             2(4-hydroxy-3,5-dimethoxyphenyl)-4-                                                                   0.061   g                                             (4-dimethyl-aminophenyl)-5-methyl-                                            imidazole-dihydrochloride                                                     methanol                1.74    g                                             H.sub.2 O               46.28   g                                             cholesterol esterase    23700   U                                             cholesterol oxidase     6500    U                                             peroxidase              230000  U                                             hexanol                 2.07    g                                             ______________________________________                                    

is prepared for the production of a reagent film in order to quantifyHDL cholesterol.

The dispersion is applied as a layer of 300 μm thickness onto apolycarbonate foil and dried with warm air. The reagent coating whichresults forms a graded blue colouration with fluids containingcholesterol depending on the cholesterol content.

    ______________________________________                                                     remission R % (measurements                                      cholesterol  in the reflection                                                concentration                                                                              photometer Reflotron.sup.R)                                      ______________________________________                                         0           70.0                                                             20           43.0                                                             40           29.0                                                             60           22.0                                                             80           18.5                                                             100          16.0                                                             ______________________________________                                    

EXAMPLE 5

Fibre mesh

Mixture of borosilicate glass fibres with a fibre diameter of ca. 0.6 μmand cellulose fibres with a fibre diameter of ca. 4 μm, preferably inthe ratio of 9:1. Weight per unit area ca. 25 g/m² at a mesh thicknessof ca. 0.21 mm; ability of the mesh to take up water of ca. 370 g/m².

EXAMPLE 6

Production of a rapid diagnostic agent (11) for the determination of HDL

If the test construction of FIG. 2 is chosen, the following measurementsapply for the different layers:

12:100×6 mm

13:5×6 mm (borosilicate fleece according to DE-A-3029579, weight perunit area ca. 60 g/m²)

14:6×6 mm

15:16×6 mm

16/17:15×6 mm

This embodiment is suitable for a sample volume between 28-32 μl and isparticularly advantageous.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

We claim:
 1. Method for determining high density lipoprotein (HDL)cholesterol in a lipoprotein-containing body fluid sample, comprisingcontacting said lipoprotein-containing body fluid sample to a firstporous carrier which contains an agent which precipitates non highdensity lipoproteins but not HDLs, so as to precipitate non-HDLs fromsaid lipoprotein containing body fluid sample, thencontacting said bodyfluid sample to a second porous carrier which retains precipitatednon-HDLs but is porous to said body fluid sample, and wherein total timerequired for both said contacting to said first porous carrier and tosaid second porous carrier is within a time period of one minute orless, and determining cholesterol in said body fluid sample as a measureof HDL cholesterol in said body fluid sample.
 2. Method for separatingnon high density lipoproteins (non-HDLs) from a lipoprotein-containingbody fluid sample, comprising contacting said lipoprotein-containingbody fluid sample to a porous carrier, wherein said carrier contains anagent which precipitates non-HDLs but does not precipitate high densitylipoproteins (HDLs), and thenseparating fluid containing precipitatednon-HDLs from said carrier and contacting said fluid to a second porouscarrier which permits a flow-through of said fluid but retainsprecipitated non-HDLs, and wherein total time required for both saidprecipitation on said first porous carrier and said separation in saidsecond porous carrier is within a time period of one minute or less. 3.Method of claim 2, wherein said second porous carrier contains acholesterol determining agent.
 4. Method of claim 2, wherein said firstporous carrier comprises a fiber fleece having a weight per unit area offrom 10 to 100 g/m² and a water absorbing capacity of 25 to 100 g/m² ata thickness of from 0.030 to 0.150 mm.
 5. Method of claim 4, whereinsaid fleece comprises fibers having a diameter of from 3 to 100 um. 6.Method of claim 4, wherein the fibers of said fiber fleece comprisefibers selected from the group consisting of synthetic resin fibers,natural fibers and glass fibers.
 7. Method of claim 2, wherein saidprecipitating agent comprises a combination of (i) a polyanion selectedfrom the group consisting of phosphotungstic acid, heparin and dextransulphate, and (ii) a bivalent cation selected from the group consistingof Mg²⁺, Mn²⁺, and Ca²⁺.
 8. Method of claim 7, wherein said polyanion isdextran sulphate having a molecular weight of about 50,000 g/mol, andsaid bivalent cation is Mg²⁺ which is present in the form of magnesiumacetate.
 9. Method of claim 2, wherein said second porous carriercomprises a fiber mesh, the fibers of which have a weight per unit areaof from 20 to 50 g/m², and water absorbing capacity of 250-500 g/m² at athickness of from 0.19 to 0.23 mm.
 10. Method of claim 2, wherein saidfirst porous carrier comprises a hydrophilic membrane having a thicknessof from 20-250 um and a pore size of from 0.2 to 20 um.